UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  4,  No.  4,  pp.  1  13-120  April  5,  1919 


FURTHER  STUDIES  ON  THE  DISTRIBUTION 
AND  ACTIVITIES  OF  CERTAIN  GROUPS  OF 
BACTERIA  IN  CALIFORNIA  SOIL  COLUMNS. 


BY 

CHARLES  B.  LIPMAN 


More  than  six  years  ago  I  prepared  for  publication,  and  soon 
thereafter  published1  some  results  of  certain  of  my  studies  on  the 
bacteria  of  California  "soil  columns."  In  that  paper  I  pointed  out 
that  by  dint  of  the  somewhat  laborious  Remy  method  I  concluded  that 
"arid"  soils  behave  differently  from  "humid"  soils  in  respect  to 
certain  groups  of  their  bacterial  flora,  in  that  among  other  things 
active  bacterial  growth  seems  to  go  on  at  relatively  great  depths  in 
the  first  class  of  soils  and  only  at  shallow  depths  in  the  second.  Much 
has  transpired  since  that  statement  appeared  which  has  contributed 
to  extensive  changes  in  science  in  general  and  in  soils  science  in  par- 
ticular. Positive  facts  and  logic  have  been  presented  which  have 
shaken  to  their  foundations  some  of  the  most  dearly  cherished  and 
firmly  established  concepts  of  a  former  generation.  The  fate  of  vast 
accumulations  of  data  on  soils  hangs  in  the  balance,  if,  indeed,  their 
doom  is  not  already  sealed.  All  of  that  has  perforce  altered  or  is 
altering  the  objectives  of  soil  work  generally,  and  has  exerted  a  pro- 
found influence  on  the  determination  regarding  the  validity  and 
significance  of  the  plans,  purposes,  and  results  of  the  investigations 
from  which  the  present  study  emanated. 

To  introduce  properly  and  to  justify  what  follows,  if  such  justi- 
fication be  needed,  it  may  not  be  amiss  to  make  brief  reference  to 
what  might  be  termed  the  parent  investigations  from  which  the 
bacteriological  studies  in  question  sprang,   and  particularly  of  the 


i  Lipman,  C.  B.,  The  distribution  and  activities  of  bacteria  in  soils  of  the  arid 
region,  Univ.  Calif.  Publ.  Agri.  Sci.,  vol.  1,  pp.  1-20,  1912. 


114  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

present  status  of  their  results  and  the  effect  of  these  results  on  the 
investigations  described  in  this  paper. 

For  many  years,  and  especially  during  the  last  fifteen  years  of 
their  lives,  the  late  Professors  Hilgard  and  Loughridge  carried  out 
mechanical  and  chemical  analyses  on  what  they  termed  the  soil 
columns  of  California.  These  soil  columns,  which  are  now  represented 
in  their  entirety  in  our  collections,  were  series  of  soil  and  subsoil 
samples  of  what  Hilgard  and  Loughridge  regarded  as  the  most  typical 
soil  classes  in  the  state  and  particularly  those  typifying  the  arid  soil 
conditions.  To  obtain  these  samples  a  type  of  post-hole  auger,  manu- 
factured by  Iwan  Brothers,  at  South  Bend,  Indiana,  was  employed, 
with  special  extensions  made  for  our  use  in  boring  at  considerable 
depths.  A  sample,  representing  an  average  of  every  foot  in  depth, 
was  usually  taken  down  to  and  including  the  twelfth  foot  wherever 
ground  water,  thick  hardpan,  or  similar  obstacles  did  not  interfere. 
If  such  obstacles  were  encountered  the  samples  were  taken  as  deeply 
as  possible.  Thus,  while  in  most  cases  the  columns  represented  twelve 
feet  of  soil  in  depth  there  were  a  number  representing  only  four,  six, 
or  seven  feet.  The  mechanical  analyses  were  carried  out  by  the  Hil- 
gard elutriator  method,  and  the  chemcial  analyses  by  the  Hilgard 
strong-acid  digestion  method.  The  ultimate  object  of  this  work  was 
to  construct  a  map  showing  the  important  soil  classes  of  California 
as  then  regarded,  and,  by  giving  information  on  the  mechanical  and 
chemical  constitution  of  the  soils,  to  enable  the  farmer  to  understand 
the  physical  and  chemical  suitability,  or  incompatibility  of  his  soil 
for  given  crops,  as  well  as  their  dependence  on,  or  independence  of 
fertilization. 

To  render  his  data  more  complete,  and  incidentally  to  throw  some 
light  on  the  microorganisms  of  arid  soils,  which  at  that  time  was  prac- 
tically an  untouched  field,  Hilgard  invited  the  writer  more  than  a 
decade  ago  to  begin  a  study  of  the  bacteria  of  arid  soils  and  to  include 
in  such  study  the  soil  columns  of  California.  Recent  studies  in  my 
laboratory,  carried  out  by  one  of  my  associates,  Dr.  D.  D.  Waynick, 
have  shown  what  we  have  been  suspecting  for  the  last  four  or  five 
years,  the  hopelessness  of  Hilgard  and  Loughridge 's  plan.  The 
reasons  for  this  may  be  briefly  stated  as  follows: 

1.  Soils  are  so  markedly  variable  that  a  column  collected  in  one 
spot  cannot  be  considered  representative  of  anything.  Even  a  compo- 
site sample  of  truly  representative  nature  is  practically  impossible 
to  obtain. 


1919]  Lipman:  Bacteria  in  California  Soil  Columns  115 

2.  A  mechanical  analysis  gives  no  idea  of  the  actual  arrangement 
of  the  soil  particles  in  situ;  hence  it  is  only  the  crudest  kind  of  guide 
to  the  soil's  physical  characters. 

3.  The  chemical  analyses  were  carried  out  by  an  arbitrary  method ; 
but  this  in  itself  would  not  be  so  serious  if  any  true  correlation  had 
ever  been  made  between  a  soil's  chemical  composition  and  its  crop- 
producing  power.  This  has  thus  far  not  been  done ;  besides,  the 
analytical  method  in  question  was  perhaps  the  least  likely  to  figure 
in  such  a  correlation  if  it  should  be  attained.  Moreover,  the  first  point 
made  above  wholly  negatives  the  validity  and  utility  of  analytical 
results  obtained  on  such  samples  and  by  such  methods  as  those  under 
consideration. 

Necessarily  these  fallacies  were  bound  to  affect  the  bacteriological 
studies,  and  many  of  the  results  as  well  as  those  of  the  chemical  and 
mechanical  analyses  are  now  resting  in  the  obscurity  which  they 
deserve.  However,  one  outcome  at  least  of  the  bacteriological  studies 
possesses  a  scientific  and  perhaps  also  a  practical  value ;  that  is,  the 
determination  of  the  depths  to  which  microorganisms  penetrate  in  arid 
soils  and  at  which  they  are  probably  active.  As  pointed  out  in  the 
opening  lines  of  this  paper,  some  of  the  results  obtained  in  work  on 
that  problem  have  already  been  published.  Further  results  would  have 
been  published  if  the  method  involved  had  not  been  so  laborious. 
Recently,  however,  we  have  found  by  a  careful  investigation  that  for 
all  practical  purposes  the  auger-collected  samples  are  just  as  good 
in  every  way  as  those  collected  by  the  special  and  laborious  method 
which  I  devised  ten  years  ago.  The  results  of  comparative  tests  of 
these  two  methods  are  given  in  a  recent  paper2  by  D.  E.  Martin  and 
the  writer.  The  fact  that  the  auger-collected  samples  are  just  as 
reliable  as  those  collected  with  special  and  great  precautions,  made 
possible  the  collection  of  ten  more  soil  columns  to  a  depth  of  six  feet, 
inclusive.  The  results  of  studies  on  these  additional  soil  columns  form 
the  chief  topic  of  this  paper,  and  are  published  primarily  for  the 
purpose  of  showing  that  bacteria  and  other  microorganisms  may 
penetrate  to  a  depth  of  at  least  seven  feet  in  most,  if  not  in  all  arid 
soils.    No  other  significance  is  claimed  for  the  data  submitted. 


2  Lipman,  C.  B.,  and  Martin,  D.  E.,  Are  the  usual  precautions  necessary  in 
taking  soil  samples  for  bacteriological  tests?  Soil  Science,  vol.  6,  no.  2,  p.  131, 
August  19,  1918. 


116  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 


Plan  of  Procedure 

The  samples  were  taken  by  Mr.  D.  E.  Martin  with  the  I  wan 
post-hole  auger.  The  detailed  method  of  sampling  is  given  in  the 
paper  cited  above.  The  samples  were  shipped  to  the  laboratory  in 
tight  Mason  fruit  jars.  The  locations  at  which  the  samples  were 
collected  are  as  follows : 

No.  1,  Roseville. — Three  hundred  yards  due  west  of  high  school; 
fifty  feet  east  of  southeast  corner  of  new  city  park ;  old  grain  field ; 
Bureau  of  Soils  classification,  San  Joaquin  sandy  loam.  Decomposed 
hardpan,  two  feet  to  two  feet  six  inches,  and  clayey  material  below. 

No.  2,  Wheatland. — One  hundred  and  fifty  yards  southwest  of 
Southern  Pacific  station;  ten  yards  west  of  highway  in  young  peach 
orchard,  on  edge  of  high  ground.  Bureau  of  Soils  classification,  Aiken 
fine  sandy  loam. 

No.  3,  Gridley. — One  hundred  yards  northwest  of  Southern  Pacific 
station ;  twenty  yards  north  of  main  street ;  middle  of  block  in  vacant 
lot;  water  table  at  five  feet.  Bureau  of  Soils  classification,  Hanford 
sandy  loam. 

No.  4,  Marysville. — One  hundred  yards  east  of  highway;  one-half 
mile  south  of  Marysville  viaduct;  Yuba  River  bottomland;  ten  feet 
elevation;  bean  field;  highly  productive.  Bureau  of  Soils  classifica- 
tion, Columbia  silt  loam. 

No.  5,  Grass  Valley. — From  apple  orchard ;  head  of  Auburn  Street 
(north  end),  Grass  Valley;  residual  from  granite.  Bureau  of  Soils 
classification,  Sierra  clay  loam. 

No.  6,  Davis. — From  University  Farm  at  Davis.  Bureau  of  Soils 
classification,  Yolo  silt  loam. 

No.  7,  Modesto. — Twenty  yards  west  of  highway;  one  hundred 
yards  north  of  warehouse  Modesto  Fuel  Company ;  northern  outskirts 
of  town  of  Modesto.    Fresno  fine  sandy  loam,  brown  phase. 

No.  8,  Fresno. — Southeast  corner  of  Harvey  and  Blackstone  ave- 
nues, East  Fresno;  vacant  lot.  Bureau  of  Soils  classification,  Madera 
sandy  loam. 

No.  9,  San  Gabriel. — Ten  yards  east  of  San  Gabriel  highway; 
one-half  mile  north  of  junction  with  El  Monte  Road  (Valley  Boule- 
vard) ;  strawberry  patch.  Bureau  of  Soils  classification,  Hanford  fine 
sandy  loam. 

No.  10,  Pasadena. — Twenty  yards  west  of  Orange  Grove  Avenue; 
ten  yards  south  of  Pasadena  Avenue  (southwest  corner);  orange 
grove.     Bureau  of  Soils  classification,  Placentia  sandy  loam. 


1919]  Lipman:  Bacteria  in  California  Soil  Columns  117 

The  tests  made  on  the  soils  consisted  of  the  usual  ammonification, 
nitrification,  and  nitrogen  fixation  determinations,  with  no  pretense 
at  attaching  importance  to  the  absolute  values  obtained.  All  the  tests 
were  made  in  the  well  known  soil  cultures  in  tumblers.  For  the 
ammonification  tests,  one  gram  of  dried  blood  was  mixed  with  fifty 
grams  of  soil  and  incubated  for  seven  days  under  optimum  tempera- 
ture and  moisture  conditions.  For  the  nitrification  tests,  one  hundred 
grams  of  soils  were  used  in  every  culture,  the  cultures  being  arranged 
in  three  ways,  viz.,  soil  alone,  soil  plus  1%  dried  blood,  soil  plus  .2% 
ammonium  sulphate.  The  incubation  period  was  one  month  under 
the  usual  conditions  of  moisture  and  temperature.  For  the  nitrogen 
fixation  test,  fifty-gram  portions  of  soil  were  employed  with  1%  of 
mannite.  The  incubation  period  was  three  weeks,  at  optimum  moist- 
ure and  temperature  conditions.  The  results  of  these  tests  are  given 
in  the  accompanying  tables.  Only  averages  of  the  determinations  are 
given  in  the  tables,  for  two  reasons.  In  the  first  place,  the  duplicates 
agreed  closely  in  most  cases ;  in  the  second  place,  no  special  im- 
portance, as  already  indicated  above,  needs  to  be  attached  for  our 
purposes  to  the  absolute  figures.  For  simplicity  and  convenience  we 
shall  discuss  briefly  each  table  separately. 


The  Ammonification  Eesults 

The  data  obtained  and  given  in  table  1  speak  for  themselves. 
There  is  every  indication  in  them  that  ammonia-producing  organisms, 
including  both  bacteria  and  fungi  are  active  at  considerable  depths  in 
all  the  soils.  Indeed,  there  is  little  indication  in  our  results  that  the 
ammonifying  activities  of  the  soils  studied  are  inferior  below  six  feet 
to  those  above  six  feet.  That  the  uniformly  high,  bacterial  efficiency 
at  ammonia  production  is  not  the  result  of  contamination  of  one  soil 
layer  by  another  had  already  been  proved  in  the  paper  above  cited,2 
and  is  proved  again  in  the  nitrification  table  accompanying  this  paper. 
In  other  words,  we  seem  to  be  justified  in  accepting  as  definitely 
proved  the  fact  that  microorganisms,  in  arid  soils  do  penetrate  to 
considerable  depths;  particularly  is  this  true  of  the  ammonia- 
producing  organisms,  which  in  the  soils  here  studied  show  about  as 
great  an  efficiency  in  the  sixth  as  in  the  first  foot  in  depth. 


118  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


The  Nitrification  Results 

A  study  of  table  2  reveals  at  once  the  great  differences  character- 
izing- the  ammonifying  and  nitrifying  powers  of  the  ten  soils  studied 
in  this  investigation,  and  especially  below  the  first  foot  in  depth. 
While  it  is  impossible  in  table  1  to  find  indisputable  evidence  that 
the  ammonifying  power  of  any  of  the  soils  decreases  downward  from 
the  surface  foot,  the  evidence  is  more  than  ample  in  regard  to  nitri- 
fication in  table  2.  In  practically  every  case  the  surface  foot  of  soil 
is  not  only  superior  but  usually  far  superior  to  those  below  it  in 
efficiency  at  nitrate  production.  The  contrast  is  indeed  very  striking. 
The  data  help,  moreover,  in  proving  that  the  method  of  sampling 
employed  is  justifiable  and  valid.  Despite  all  this,  however,  table  2 
gives  unquestionable  evidence  in  support  of  the  idea  that  even  nitri- 
fying organisms  do  penetrate  as  far  down  as  the  seventh  foot  of  soil 
under  arid  conditions.  While  their  work  may  be  feeble  in  many  arid 
soils  at  considerable  depths  it  is  quite  vigorous  in  many  others  at 
similar  depths.  Such  activity  is  manifested  regardless  of  the  form 
of  nitrogen  available  for  nitrification.  As  a  rule,  it  appears  that  the 
more  fertile  soils,  like  those  at  Gridley  and  Davis,  are  those  in  which 
bacterial  activity  is  greatest  in  the  deeper  layers.  Owing  to  the 
organic  matter  supply  in  such  soils,  contrasted  with  that  in  the  poorer 
soils,  like  those  at  Fresno  and  Modesto,  it  is  natural  that  the  bacterial 
efficiency  should  vary  accordingly,  which,  indeed,  is  just  as  true  in  the 
surface  foot  of  soil  as  in  the  deeper  layers. 


The  Nitrogen  Fixation  Results 

The  nitrogen  fixation  results  are  given  in  table  3.  They  cannot 
be  regarded  as  being  of  much  significance,  owing  to  the  considerable 
error  which  attaches  to  a  nitrogen  determination  on  a  ten-gram  por- 
tion of  soil.  The  determinations  were  made  in  duplicate,  but  only 
the  averages  are  given  in  the  table ;  these  represent  five  times  the 
quantity  of  nitrogen  found  in  ten  grams  of  the  soil  culture,  minus 
the  amount  found  in  the  sterile  control  portion  taken  for  analysis. 
It  is  easy  to  see  that  such  manipulation  may  easily  lead  to  serious 
errors.  However  that  may  be,  the  results  are  given  for  whatever 
interest  rind  value  the}'  may  possess.  In  the  case  of  the  San  Gabriel 
soil,  it  is  probably  true  that  the  figures  represent  actual  gains  in 
aitrogen,  and  it  is  interesting  to  note  that  while  nitrogen  fixation  in 


1919]  Liyman:  Bacteria  in  California  Soil  Columns  119 

this  soil  is  less  in  the  lower  than  in  the  upper  layers,  it  seems  to  be 
quite  definite.  From  this  soil,  at  least,  it  would  appear  that  the 
nitrogen  fixation  results  are  in  general  accord  with  the  ammonification 
and  nitrification  results  regarding  the  chief  point  made  in  this  paper, 
viz.,  that  bacterial  life  does  extend  into  the  deeper  layers  of  the  soil 
under  arid  conditions.  Through  an  oversight  the  Pasadena  soil  was 
not  studied  in  regard  to  nitrogen  fixation,  hence  no  statement  appears 
with  regard  thereto  in  table  3. 

Summary  and  Conclusion 

From  studies  on  twelve  soils,  two  of  which  are  discussed  in  another 
paper  and  ten  in  this  paper,  the  writer  has  been  able  to  confirm  his 
findings  of  several  years  ago  to  the  effect  that  microorganisms  of  arid 
soils  penetrate  deeply  into  the  subsoil  layers. 

In  the  present  paper  it  has  been  demonstrated  that  ammonifying 
vigor  continues  undiminished  through  six  feet  of  soil  in  every  case. 
While  this  is  not  true  for  the  nitrifying  powers  of  the  same  soils,  it 
is  clear  that  nitrifying  bacteria  do  live  in  the  lower  layers  of  some,  and 
perhaps  of  all  of  the  soils  to  similar  depths.  The  great  difference 
between  the  two,  however,  lies  in  the  fact  that  the  nitrifying  power  of 
these  soils  diminishes  rapidly  downward  from  the  first  foot  while  the 
ammonifying  power  remains  apparently  unchanged.  The  nitrogen 
fixation  results  are  for  the  most  part  inconclusive,  but  such  as  are 
above  question  seem  in  general  to  confirm  the  idea  that  bacteria  and 
other  microorganisms  do  penetrate  to  greater  depths  in  arid  soils  than 
one  would  expect.  Such  penetration  and  activity  at  those  depths  seem 
to  be  superior  to  those  noted  on  humid  soils,  so  far  as  we  have  evidence 
in  the  literature  upon  which  to  base  such  a  comparison. 

Table  1. — Ammonification 
Milligrams  of  Nitrogen  as  Ammonia  Produced 
Depth  in  feet 

f A ^ 

Name  of  soil  12  3  4 

Eoseville   30.84  31.89  29.17  10.00 

Wheatland    35.84  39.13  32.60  28.27 

Gridley    38.22  32.20  36.96  30.80 

Marysville    33.81  34.79  35.28  38.65 

Grass  Valley ....  23.10  24.50  20.30  22.96 

Davis  68.18  54.88  44.94  37.66 

Fresno  38.71  38.64  37.03  36.54 

Modesto 38.71  42.63  40.18  39.48 

San  Gabriel 42.14  38.57  50.22  38.36 

Pasadena    56.35  52.64  46.34  52.71 


5 

22.12 

6 
20.79 

28.00 

22.96 

22.82 

14.49 

36.33 

36.96 

23.94 

21.63 

40.60 

41.58 

33.74 

30.52 

42.14 

41.40 

37.24 

32.34 

41.93 

18.18 

120 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


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